Probing Rad51-DNA interactions by changing DNA twist

Atwell, Scott; Disseau, Ludovic; Stasiak, Alicja Z.; Stasiak, Andrzej; Renodon-Cornière, Axelle; Takahashi, Masayuki; Viovy, Jean‐Louis; Cappello, Giovanni

doi:10.1093/nar/gks1131

Cited by 19 publications

(21 citation statements)

References 40 publications

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“…We use Ca 2+ as the divalent cation as it inhibits ATP hydrolysis and thereby prevents protein dissociation, resulting in the formation of stable filaments (33,34). Assembly was monitored both by tracking the length increase of the DNA-tether determined from the beads’ z -position and by observing unwinding of the DNA determined by tracking the rotation angle about the DNA-tether axis [Figure 1b; note that in the FOMT configuration we determine the rotation angle by simply converting the tracked ( x , y )-position on the circular annulus of the bead’s free rotation and converting it to angle, see Supplementary Figure S1] (26,29).…”

Section: Resultsmentioning

confidence: 99%

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Structural and torsional properties of the RAD51-dsDNA nucleoprotein filament

Lee

Lipfert

Sánchez

et al. 2013

Nucleic Acids Research

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Human RAD51 is a key protein in the repair of DNA by homologous recombination. Its assembly onto DNA, which induces changes in DNA structure, results in the formation of a nucleoprotein filament that forms the basis of strand exchange. Here, we determine the structural and mechanical properties of RAD51-dsDNA filaments. Our measurements use two recently developed magnetic tweezers assays, freely orbiting magnetic tweezers and magnetic torque tweezers, designed to measure the twist and torque of individual molecules. By directly monitoring changes in DNA twist on RAD51 binding, we determine the unwinding angle per RAD51 monomer to be 45°, in quantitative agreement with that of its bacterial homolog, RecA. Measurements of the torque that is built up when RAD51-dsDNA filaments are twisted show that under conditions that suppress ATP hydrolysis the torsional persistence length of the RAD51-dsDNA filament exceeds that of its RecA counterpart by a factor of three. Examination of the filament’s torsional stiffness for different combinations of divalent ions and nucleotide cofactors reveals that the Ca2+ ion, apart from suppressing ATPase activity, plays a key role in increasing the torsional stiffness of the filament. These quantitative measurements of RAD51-imposed DNA distortions and accumulated mechanical stress suggest a finely tuned interplay between chemical and mechanical interactions within the RAD51 nucleoprotein filament.

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Section: Resultsmentioning

confidence: 99%

“…During filament assembly, the magnet was rotated by ∼−310 turns (34) to relieve the accumulated torsional strain. Subsequently, the extension of the RAD51 filament (Figure 2b) and the torque stored in the filament (Figure 2c) were simultaneously measured as a function of turns.…”

Section: Resultsmentioning

confidence: 99%

Structural and torsional properties of the RAD51-dsDNA nucleoprotein filament

Lee

Lipfert

Sánchez

et al. 2013

Nucleic Acids Research

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“…Similar conformational transitions have also been observed for hRAD51 filaments on dsDNA. Experiments using magnetic tweezers (Ristic et al, 2005;Atwell et al, 2012) have shown that hRAD51 binds dsDNA in two distinct modes with different pitch and that, upon ATP hydrolysis, the NPFs can switch to a compact state before NPF disassembly. Changes in DNA twist can also trigger transitions between the two modes.…”

Section: Introductionmentioning

confidence: 99%

Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

et al. 2018

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An essential mechanism for repairing DNA double‐strand breaks is homologous recombination (HR). One of its core catalysts is human RAD51 (hRAD51), which assembles as a helical nucleoprotein filament on single‐stranded DNA, promoting DNA‐strand exchange. Here, we study the interaction of hRAD51 with single‐stranded DNA using a single‐molecule approach. We show that ATP‐bound hRAD51 filaments can exist in two different states with different contour lengths and with a free‐energy difference of ~4 kBT per hRAD51 monomer. Upon ATP hydrolysis, the filaments convert into a disassembly‐competent ADP‐bound configuration. In agreement with the single‐molecule analysis, we demonstrate the presence of two distinct protomer interfaces in the crystal structure of a hRAD51‐ATP filament, providing a structural basis for the two conformational states of the filament. Together, our findings provide evidence that hRAD51‐ATP filaments can exist in two interconvertible conformational states, which might be functionally relevant for DNA homology recognition and strand exchange.

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“…HR is a process that requires the presence of a homologous DNA region to serve as a template for accurate repair (2). RAD51, a recA homolog that forms helical filaments, binds to single-strand DNA, thus promoting recombinational repair (3,4). RAD51 knockout mice usually die at an early stage of embryogenesis, and cells lacking functional RAD51 often exhibit chromosome breaks and cell-cycle arrest (5,6).…”

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confidence: 99%

Regulation of homologous recombinational repair by lamin B1 in radiation‐induced DNA damage

et al. 2015

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DNA double-strand breaks (DSBs) are the major lethal lesion induced by ionizing radiation (IR). RAD51-dependent homologous recombination (HR) is one of the most important pathways in DSB repair and genome integrity maintenance. However, the mechanism of HR regulation by RAD51 remains unclear. To understand the mechanism of RAD51-dependent HR, we searched for interacting partners of RAD51 by a proteomics analysis and identified lamin B1 in human cells. Lamins are nuclear lamina proteins that play important roles in the structural organization of the nucleus and the regulation of chromosome functions. Immunoblotting analyses revealed that siRNA-mediated lamin B1 depletion repressed the DNA damage-dependent increase of RAD51 after IR. The repression was abolished by the proteasome inhibitor MG132, suggesting that lamin B1 stabilizes RAD51 by preventing proteasome-mediated degradation in cells with IR-induced DNA damage. We also showed that lamin B1 depletion repressed RAD51 focus formation and decreased the survival rates after IR. On the basis of these results, we propose that lamin B1 promotes DSB repair and cell survival by maintaining the RAD51 protein levels for HR upon DSB induction after IR.-Liu, N.-A., Sun, J., Kono, K., Horikoshi, Y., Ikura, T., Tong, X., Haraguchi, T., Tashiro, S. Regulation of homologous recombinational repair by lamin B1 in radiation-induced DNA damage. FASEB J. 29, 2514-2525 (2015). www.fasebj.org Key Words: B-type lamins • nuclear envelope • progeria syndrome • RAD51 • recombinational DNA repair AMONG THE VARIOUS TYPES of DNA damage induced by ionizing radiation (IR), double-strand breaks (DSBs) are regarded as the most serious impairment leading to cell death. In mammalian cells, DSBs are repaired mainly by 1 of 2 genetically distinct processes, nonhomologous end joining and homologous recombination (HR) (1). HR is a process that requires the presence of a homologous DNA region to serve as a template for accurate repair (2). RAD51, a recA homolog that forms helical filaments, binds to single-strand DNA, thus promoting recombinational repair (3, 4). RAD51 knockout mice usually die at an early stage of embryogenesis, and cells lacking functional RAD51 often exhibit chromosome breaks and cell-cycle arrest (5, 6). RAD51-defective animals and cells are both sensitive to IR. On the other hand, RAD51 overexpression in different organisms and cell types apparently increases genomic instability by stimulating aberrant recombination between short repetitive elements (7,8). These findings indicate that RAD51 is a critical protein in HR repair and cell survival. Interestingly, RAD51 has been shown to form nuclear foci at sites containing DSBs (9, 10). However, the mechanisms that regulate the RAD51 protein levels and its focus formation in response to DNA damage remain unclear.Higher-order nuclear architectures, including the nuclear lamina, are considered to be essential for the proper regulation of DNA metabolism, including gene activation and silencing, as well as DNA replication and ...

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Probing Rad51-DNA interactions by changing DNA twist

Cited by 19 publications

References 40 publications

Structural and torsional properties of the RAD51-dsDNA nucleoprotein filament

Structural and torsional properties of the RAD51-dsDNA nucleoprotein filament

Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

Regulation of homologous recombinational repair by lamin B1 in radiation‐induced DNA damage

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